RDCs 10.8

NMR of Biological Macromolecules in Solution More resonances; shorter T2/broader lines Similar basic techniques- HSQC, TOCSY, NOESY Other experiments built off of INEPT/DEPT/TOCSY/NOESY 3D or more dimensions to improve resolution Use 2D HSQC like chemists use 1D 1H Higher magnetic field to improve resolution Isotopic labeling for signal-to-noise Cryo probes for signal-to-noise 4-channel consoles- 1H/13C/15N/2H or 1H/13C/31P/2H NMR Technique development

xDNA

NOESY Liu, Kool, et al., Science, 2003: 302., pp. 868 – 871; Liu, Kool, J. Am. Chem. Soc., 126 (22), , Lynch, Kool, et al., J. Am. Chem. Soc., 128 (45), , 2006.

3D NMR and Beyond What do you do when resonances are overlapped in 2D? => 3D NMR The concept of 3D NMR is essentially the same as 2D, except now there are 2 indirect detect dimensions, so 2 times (now t1 and t2) that get incremented to give a second and third dimension FID Of course, what do you do when resonances are overlapped in a 3D experiment, you acquire a 4D experiment…. The main application of 3D and 4D NMR is structural biology, using NMR to determine 3D structures of macromolecules, primarily proteins and nucleic acids. High-resolution 3D structures of molecules of ~50, ,000 MW have been determined with NMR.

3D NMR and Beyond 1) Ligand-macromolecule interactions The limit of the largest structure of a macromolecule structure determined by NMR is always increasing. Even when a high-resolution structure is not realistic, NMR assignments can be made on the protein backbone or the ligand. 3D NMR and 4D NMR is required for the protein, 3D NMR can also be very useful although not necessarily required on the ligand. 2) Larger small molecules can have overlapped protons, even in 2D NMR, especially molecules with somewhat repetitive units like a sugar.

Difficulties with 3D data 1) Difficult to display 2) Data sets are disk space intensive (>100 mb - 1 gb) 3) Data processing is challenging, hard to process and very time consuming. 4) Data acquisition takes several days of NMR time. 5) Pulse sequences written have limited use and are somewhat difficult to optimize 6) Experiments depend upon full isotope labeling of 13 C and/or 15 N. 7) More dimensions on an experiment, longer pulse sequence, T2 relaxation kills signal.

What experiments exist in 3D? 2D NMR can be broken down into through-bond and through-space 3D NMR can be broken down into through-bond, through-space, through-bond- through-space/through-space-through-bond

Through-Space-Through-Bond 1) NOESY-HSQC (first NOESY then an HSQC) (there are also ROESY-HSQC or NOESY-HMQC...) Proton-proton NOESY with one of the proton dimensions correlated to a Carbon or Nitrogen. Often referred to as a 3D NOESY or as a 13 C-edited NOESY (a NOESY edited by spreading proton NOEs over a third dimension 13 C or 15 N)

NOESY-HSQC

2D NOESY vs. 3D NOESY-HSQC ( 15 N) on a protein. 2D NOESY 3D NOESY-HSQC ( 15 N)

Through-Bond-Through-Space 2) HSQC-NOESY Approximately the same as the NOESY-HSQC. The HSQC dimension is first rather than the NOESY. 1st indirect dimension is 1 H, then the 13 C (or 15 N), then there is an NOE mixing time then detect.

Through-Bond-Through-Space 3) TOCSY-NOESY First 1 H- 1 H TOCSY, then NOESY For example- suppose proton H5 has identical chemical shift to H5*, but H6 has a different chemical shift than H6*, in 2D NOESY it was established that H5 or H5* has NOE to H A. First, TOCSY: H6 has TOCSY crosspeak with H5, H6* with H5*. then NOESY, H5 has NOE to H A, H5* does not. The NOE has been discriminated based upon the chemical shift of H6 and H6* At chemical shift of H6, H5, H A : there will be a crosspeak but not at H6*, H5*, H A

Through-Bond Through-Bond can be broken into two categories: experiments with a diagonal (like a COSY or TOCSY in 2D) and experiments without a diagonal (like an HSQC in 2D) Experiments with a diagonal: 1) HSQC-TOCSY First, an HSQC correlating the proton and nitrogen or carbon, then a TOCSY. Similar to a NOESY-HSQC When there are resonances with coincidental chemical shifts in proton but not carbon, you can acquire a 3D HSQC-TOCSY and determine which resonance is which. This can be acquired as 2D carbon/proton as well.

HSQC-TOCSY First, HSQC, record 13 C shift Then, TOCSY, detect proton

2) HCCH TOCSY (a 13 C- 13 C TOCSY) Similar to HSQC-TOCSY in that it is an HSQC first then a TOCSY, except it uses the large 13 C- 13 C coupling for the TOCSY. Must be 13 C labeled though. 13 C- 13 C coupling is Hz so signal-noise is much better than in standard proton TOCSY, There is no torsion angle dependence, so correlations can be observed even when proton-proton three bond scalar coupling is 0 Hz, e.g. when the torsion angle between 2 protons is ~90 .

First, transfer magnetization from proton to carbon as in HSQC Then, do 13 C- 13 C TOCSY Then, transfer magnetization back to proton and detect

Through-Bond Experiments without a diagonal Essentially, these experiments are developed to have one or two correlations per repeating unit to assign the proton spectrum. Many of these can be acquired as 2D experiments. Many require either 13 C or 15 N labeling, but some can certainly done in natural abundance especially with a cryo probe at 900 MHz DNA/RNA 1) HCNCH/HCN 2) Exchangeable Correlations of RNA/DNA base 3) Backbone HCP Correlation Proteins Backbone correlations

Assignment of NMR Spectra of Macromolecules Proteins: Protein assignments depend primarily upon 3D experiments correlating 1 H, 13 C and 15 N: Normally, one starts with the amide proton-nitrogen correlation (HSQC), as the amide proton and nitrogen are common to all amino acids except proline and well dispersed in proteins.

Protein Backbone Correlations- Protein Assignments

HNCA

Nucleic Acids Usually, you start assignments with the protons on the base of the nucleotide and the anomeric proton (H1') as those are the most dispersed in chemical shift. HCN Magnetization is transferred from H8 to C8 to N9 and from H1' to C1' to N9. Correlations are observed between H8 and N9 and H1' and N9 in 2D (in 3D H1', C1', N9 and H8, C8, N9). There are many different versions of this sequence and the base to sugar correlation.

Nucleic Acids HCCH TOCSY Magnetization is transferred from H1' to C1' (also H2' to C2'...) then C1' to C2', C3' C4', C5' then from C2' to H2', C3' to H3'... Correlations are observed H1'/C1'/H2',H3',H4',H5',H5"; H2'/C2'/H1',H3',H4',H5',H5"...

Nucleic Acid Backbone Correlations ( 1 H/ 13 C/ 31 P) 3D HCP Two ( 1 H/ 31 P) or three dimensional ( 1 H/ 13 C/ 31 P) experiment similar to HNCA for proteins CorrelatesH4' - C4' - P (i) H4' - C4' - P (i+1) H5' - C5' - P (i) H5" - C5' - P (i) H3' - C3' - P (i+1) H2' - C2' - P (i+1)

Correlates H5' - P (i) H5" - P (i) H3' - P (i+1) (H4' - P (i)) 2D 1 H/ 31 P Heteronuclear COSY (or HP-COSY)

Dipole-Dipole Coupling Dipole-Dipole Coupling = dipolar coupling Direct interaction of two spins with the magnetic field  B  (3cos 2  - 1)/ r -3  B is the splitting induced by the magnetic field or dipolar coupling  is the angle between the vector connecting the two nuclei and the magnetic field r is the distance between the two nuclei

Dipole-Dipole couplings can be 1000s of Hz Methylene Chloride

Why do you not normally observe dipole-dipole couplings?  B  (3cos 2  - 1)/ r -3 In solution, molecules normally are tumbling freely, or isotropically. In solids, molecules are not freely tumbling, and dipolar couplings are observed. Is there a way to observe them in solution? By inducing anisotropic tumbling of the molecule in solution.

Why would we want to observe the residual dipolar coupling (RDC)? Information on the orientation of bond vectors in solution or long range distance measurement between nuclei.

Residual Dipolar Couplings on Small Molecules

Residual Dipolar Coupling Measurements Coupling constants can be measured in 2D experiments in several ways, the most common being measuring the frequency difference between 2 resonances. Another method is to measure the intensity of the crosspeak of interest and compare that intensity to the diagonal peak in a specific spectrum or similarly measure the intensity of a resonance and compare to the intensity of the resonance in a reference spectrum. Measuring Heteronuclear 1 J CH / 1 J NH with an HSQC

Scalar Coupling Constants Across a Hydrogen Bond Hydrogen Bonds can be directly detected. Small scalar coupling constants have been observed and measured for nuclei involved in Hydrogen bonds. The experiments used to detect Hydrogen bonds are COSY type experiments, hetero or homonuclear. Primarily these experiments have been applied on nucleic acids, proteins and protein-nucleic acid complexes, but in principal are applicable to any molecule with Hydrogen bonds. The experiments are much more sensitive for N-HN Hydrogen bonds using 15 N/ 1 H correlations; but could use 15 N/ 15 N correlation or 1 H/ 1 H correlation for detection. Also, N-HO(-CH) or O-HO(-CH) using 1 H/ 1 H or 1 H/ 13 C correlations are possible, but weaker.

Scalar Coupling Constants Across a Hydrogen Bond J HN COSY Transfers magnetization from 1 H to 15 N (across the Hydrogen bond, uses 1 H - 15 N coupling constant across Hydrogen bond) Then back to the 1 H; What is observed is an 1 H/ 15 N HSQC with peaks at the 1 H chemical shift of the Guanine H1 and the nitrogen chemical shift of both the Cytosine N3 and Guanine N1. Cytosine Guanine

J NN COSY Transfers magnetization from 1 H to 15 N to 15 N (across the Hydrogen bond, uses 15 N - 15 N coupling constant across Hydrogen bond) then back to the 1 H. What is observed is an 1 H/ 15 N HSQC with peaks at the 1 H chemical shift of the Adenine H2 and the nitrogen chemical shift of all of the Adenine N3, Adenine N1 and Uracil N3. Uracil Adenine

TROSY TROSY = Transverse Relaxation Optimized SpectroscopY Transverse Relaxation = T2 As molecular weight increases, T2 relaxation times get shorter. As T2 gets shorter, the efficiency of through-bond correlation experiments gets worse. The problem with studying large proteins (>30,000 MW) is not just the amount of resonances and the broadening of the resonances dues to slow tumbling, but also the loss of signal in correlation experiments from T2 relaxation. The goal of TROSY is to limit the effect of T2. TROSY is another way to transfer magnetization from one nucleus to another (proton to carbon, proton to nitrogen), and can be incorporated into any of the heteronuclear correlation experiments

TROSY HSQC without decoupling in t1 or t2 Normal HSQC TROSY

1D Slice through HSQC TROSY HSQC

Applications of TROSY The TROSY effect has now been applied to all pulse sequences relevant to macromolecules. The TROSY effect is greater for larger molecules. The TROSY effect is enhanced at very high field for 15 N, and sometimes for 13 C; theoretically, 1 GHz would produce the maximal TROSY effect for 15 N/ 1 H correlations. TROSY is thus most useful on high molecular weight molecules at high field for 1 H/ 15 N correlations. An important use is thus a quick examination of protein structure. Acquire 1 H / 15 N-TROSY-HSQC on large protein, then bind small molecule drugs to see possible changes in spectra upon binding. Drugs that don't bind should cause no changes in spectra, those that bind specifically should cause dramatic changes of a few residues.